This invention is concerned with the separation of lipin-containing particles from aqueous milieu. More specifically this invention relates to the removal of lipoprotein or glycolipid-containing vesicles from aqueous suspensions. This invention is particularly concerned with the general, nonspecific adsorption of microbes such bacteria, yeast, fungi and viruses from contaminated aqueous suspensions.
Lipins are a group of compounds comprising fats and lipoids which are soluble in ether. They include fats, fatty oils, essential oils, waxes, sterols, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids, fatty acids and lipoproteins.
A great variety of biological structures contain lipins. For example, particles such as animal viruses may contain lipids at up to about 50 percent by weight. Chylomicons, a major circulating lipid transport medium in higher animals, are essentially fat globules enveloped by a lipoprotein membrane. Animal cells, bacteria, yeast and fungi all contain varying proportions of lipins in their cell walls and protoplasm.
Chylomicrons, liposomes, cellular microorganisms and animal cells are examples of lipin vesicles, a major class of lipin-containing particles with which this invention is concerned. Lipin vesicles are generically defined as substantially water insoluble particles ranging about from 250 to 10,000 A in mean diameter which are characterized by a lipin-containing membranous envelope enclosing a liquid interior. The contained liquid may consist almost entirely of lipid, as in the case of chylomicrons, or be relatively free of the substance, as in the case of microorganisms. It is not necessary that the contained liquid have any lipid content whatsoever.
The presence of such lipin particles in aqueous suspensions has presented many problems for a great variety of arts. The foremost difficulty has been encountered with microbial contamination of aqueous liquids intended for administration to living organisms, particularly parenteral fluids infused into patients. While parenteral fluids are carefully manufactured so as to be sterile it is common practice for users to add medications or other additives to the solutions. This provides a potential avenue for contagion to enter the patient's blood stream. Thus it has been previous practice to modify the parenteral solution administration sets which are used to provide a controlled fluid flow from the solution container to the patient's vein by the inclusion of a filter capable of physically entrapping cellular microorganisms. Similar filters have been used with solution administration sets in continuous ambulatory peritoneal dialysis. Such filters, hereinafter referred to as sterile filters, are generally porous membranes having an average pore diameter of about from 0.2 to 0.45 microns, ordinarily about 0.2 microns. These filters are capable of retaining most cellular microorganisms since the smallest bacterium is believed to be about 0.3 microns in diameter.
The pharmaceutical industry has also employed membrane filters of this type to sterile filter products which cannot be chemically or thermally sterilized because of their lability. Examples of such products include insulin and human blood protein fractions such as factor VIII.
Sterile filters approach the absolute in retaining particles greater than the stated pore size. However they are readily clogged by relatively small numbers of particles, particularly those which have a pore size close to the average pore size of the filter. Consequently, it is conventional to pass the liquid to be sterilized through a depth filter before contacting it with the sterile filter. These filters have a high capacity to retain particles throughout rather than by sieving only at the liquid-filter interface.
Depth filters are fabricated from many materials including cellulose, polypropylene, diatomaceous earth and asbestos. Most of the depth filters trap particles by physical entrapment at points where two or more fibers or granules form a pore, alone or in combination with what usually are assumed to be London-van der Waal's attractive forces. Depth filters have the advantage of removing particles while retaining a high filter flux, i.e., a high flow rate of feedstock per unit of filtration area, even in the face of a particle load that would rapidly clog a sterile filter. However, depth filters are largely ineffective in removing particles in the 0.5 to 3 micron size range, at least when compared to sterile filters. Of course, viruses are too small to be retained even by sterile filters. A long felt need has existed for a unitary filter which is capable of removing particles having a wide range of sizes from suspension, including particularly bacteria and viruses, without suffering a significant reduction in flux when exposed to heavy particle loads. At the very least, considerable improvement could be secured by using a depth filter having such characteristics, thus freeing the sterile filter from its role as the virtually sole line of defense against passage of the smallest cellular organisms and thereby reducing the probability of clogging or flux reductions.
The sterile filters used in parenteral administration sets rarely have to deal with high levels of suspended particles, and thus clogging is not usually encountered. However, the flux of hydrocolloidal solutions such as blood protein fractions through sterile filters is very low. This low flux is attributed to the affinity of the hydrocolloids for the filter surfaces, resulting in increased hydrocolloid binding by the filter and the formation of hydrocolloid concentration gradients upstream from the filter surface. Similar problems are encountered in industrial sterile filtration of the same products. Consequently, membrane filters used in conventional parenteral administration sets for large-volume, non-colloidal parenterals such as dextrose or protein hydrolysates in water have proven inadequate for the filtration of viscous hydrocolloid solutions such as factor VIII or albumin.
Filters having small pores create another problem in parenteral solution administration. In the low pressure environment of an infusion any residual air in the administration set tubing will accumulate against the wetted filter rather than passing through. This phenomenon is termed air-blocking, and frequently it requires that the set be discarded. Efforts have been made to remedy this problem by inclusion of a non-wetting or hydrophobic filter in the set in addition to the normal hydrophilic member (U.S. Pat. No. 4,004,587). This of course necessitates the inclusion of two different types of filters in the set, an added expense.
Finally, all filters which rely solely upon the mechanical exclusion of particles depend upon the dimensional stability of the particles. If the particles can deform so as to pass through the micron sized pores of the filter then the filter will effectively fail. Mycoplasma, which are bacteria devoid of rigid cell walls, are highly deformable. Other families of microorganisms also exhibit varying degrees of deformation under pressure, as do mammalian cells and chylomicrons. The filtration of suspensions of such particles would be more reliable if mechanical exclusion could be supplemented.
In summary therefore it is apparent that the previous efforts of the art to remove particles from suspension primarily on the basis of mechanical exclusion has resulted in considerable difficulties.
Clinical chemistry is another art in which lipin particles have created problems. Blood samples taken for diagnostic assay of constituents are generally permitted to clot and the resulting serum removed by aspiration or decantation, frequently with the aid of devices such as disclosed in U.S. Pat. No. 3,865,731. While this process removes most cellular matter from the test sample it fails to reduce the level of other insoluble particles, most notably chylomicrons. Such particles interfere in subsequent optical assays of serum constituents.
An optical assay is defined as any analytical method in which the concentration or activity of an analyte is measured by a change in light as it is passed into the sample, and includes nephelometry and spectrophotometry in the main. Lipemic serum samples often contain chylomicrons in such concentrations that the serum appears milky, and even at lower chylomicron concentrations light scattering particles in the sample will interfere. While such samples may be diluted to reduce the interference this also necessarily dilutes the analyte, thereby reducing sensitivity, and in any case the comparative effect of the chylomicrons is still significant relative to analyte concentration. Reagents such as detergents may be added to destroy the lipid suspension but may interfere in various assays (United Kingdom Pat. No. 1,542,982). Ultracentrifugation will remove the particles but requires costly equipment and is tedious to perform. A need therefore exists for a method and device to remove lipin particles from biological fluids to be assayed by optical methods.
A need also exists in many arts to nonspecifically remove animal viruses from aqueous compositions. While the virions in many aqueous substances can be inactivated by pasteurization or chemical sterilization, many labile products, particularly pharmaceuticals and some blood protein fractions, are sensitive to such harsh treatments. These techniques are also not suitable for high volume treatments such as drinking water purification because of the high cost. Mechanical entrapment of virions by filtration ordinarily is not practical because at the required pore sizes the filter flux is extremely low.
Various investigators have looked into the use of immunoadsorbents to separate hepatitis virus. However, this technique is of little use because supplies of antihepatitis are limited, there is a risk of leaching antibody into the adsorbent effluent and, primarily, the antibody is necessarily capable of binding only the hepatitis virion and is not effective in removing other harmful viruses.
Immunoadsorbents have also been used in various affinity chromatography techniques for cell separation. See Cuatrecasas et al., "Ann. Rev. Biochem." 40:275 (1971). In these techniques inert matrices are substituted with ligands. Animal cells expected to contain membrane receptor proteins for the ligands are contacted with the immobilized haptens. Those cells having receptors specific for the ligand are bound while the remainder are washed free of the substrate, thus enabling one to obtain specific cell lines. Such methods are, however, of no use where the object is to remove a diverse cell population from suspension because the receptor sites are unknown and, in any case, would be so numerous that preparing immobilized ligands for all of them would be impractical.
This handicap would appear to be shared by the process of U.K. patent specification No. 1,531,558 to Kabi. This patent discloses adsorbing hepatitis virus from plasma and some solutions of blood protein fractions with a water permeable matrix having a coupled hydrophobic ligand of more than 7 carbon atoms or a condensed ring system. The adsorbent is disclosed to have a high and specific affinity for hepatitis virus. A need therefore remains for an adsorbent for animal viruses which is not specific for any one virus.
Tanny et al., "J. Parenteral Drug Association" 33(1):40-51 (1979) speculate that 0.45 and 0.20 micron cellulose triacetate membranes retain Pseudomonas diminuta by a combined adsorptive and sieve effect. Similarly, Tanny et al. advance the same hypothesis to account for losses in the titer of influenza vaccine passed through mixed cellulose esters, cellulose triacetate and acrylonitrile-vinyl chloride copolymer ["J. Parenteral Drug Association" 32(6):258-267 (1978)].
Pertsovskaya et al. "Biol. Nauki" 14(3):1005 (1971) disclose that glass, methylene and amine-substituted glass, and films of polyamide, polysacrylate, cellulose triacetate, and polyethylene all adsorb different groups of bacteria to varying degrees. In some cases, e.g., with bacilli, no adsorption at all was observed. Gerson et al. in Immobilized Microbial Cells, K.Ven Katsubramanian, Editor, pp 39-43 (1978) also report adsorbing various bacteria to surfaces.
Ambergard.TM. filters, which are ion exchange resins having the structure R-N.sup.+ (CH.sub.3).sub.3 X wherein R is a styrenedivinylbenzene copolymer and X is OH--, Cl-- or SO.sub.4 =, have been used to upgrade the bacteriological quality of demineralized water for ultimate use in pharmaceuticals (Rhom and Haas literature dated June, 1978). In this connection, see U.K. patent application No. 2,009,623A.
U.S. Pat. Nos. 4,007,113 and 4,007,114 to Ostreicher employ a matrix of self bonding and electronegative fibers having surfaces coated with malamine-formaldehyde cationic colloid for filtering contaminated liquids.
Hjerten et al., "J. of Chromatography" 101:281-288 (1974) discloses that satellite tobacco necrosis virus and baker's yeast cells are retained in columns of non-ionogenic hydrophobic agarose in the presence of elevated salt concentrations.
Similarly, Magnusson et al. in "Immunology" 36:439-447 (Mar. 19, 1979) disclose that adsorption of S. typhimurium occurs when placed on a column in the presence of 1M (NH.sub.4).sub.2 SO.sub.4, but that the bacteria elute as the salt concentration is reduced.
Halperin et al., "Biochemical and Biophysical Research Communications" 72(4):1497-1503 (1976) disclose desorbing erythrocytes retained on alkyl agarose columns by repeated pipetation in the presence of bovine serum albumin.
Accordingly it is an object of this invention to adsorb a wide spectrum of cells including animal cells, unicellular organisms, bacteria, yeast, fungi and viruses, from aqueous suspensions, using a single adsorbent composition.
It is another object to provide improved hydrophobic adsorbent compositions.
It is a further object of this invention to remove lipin particles from lipemic body fluids such as serum or plasma and provide an improved device therefor.
It is another object to pasteurize alcoholic beverages without the cost and detriment to flavor inherent in prior methods.
It is an additional object of this invention to sterile filter parenteral solutions, particularly solutions containing protein or low concentrations of salt, at increased flux and with greater assurance of sterility than heretofore possible, and to provide an improved device therefor.
It is another object to provide an improved surface for the cultivation of mammalian cells in tissue culture or for binding enzyme-containing lipin particles used in enzyme reactors.
These and other objects of this invention will be apparent from consideration of this specification as a whole.